实验十一 ＤＮＡ的酶切

1. 实验十一ＤＮＡ的酶切

2. Restriction Endonucleases:  An Overview • Restriction enzymes were discovered about 30 years ago during investigations into the phenomenon of host-specific restriction and modification of bacterial viruses. • Bacteria initially resist infections by new viruses, and this "restriction" of viral growth stemmed from endonucleases within the cells that destroy foreign DNA molecules. Among the first of these "restriction enzymes" to be purified were EcoR I and EcoR II from Escherichia coli, and Hind II and Hind III from Haemophilus influenzae. These enzymes were found to cleave DNA at specific sites, generating discrete, gene-size fragments that could be re-joined in the laboratory. • Researchers were quick to recognize that restriction enzymes provided them with a remarkable new tool for investigating gene organization, function and expression. As the use of restriction enzymes spread among molecular biologists in the late 1970’s, companies such as New England Biolabs began to search for more. Except for certain viruses, restriction enzymes were found only within prokaryotes. • Many thousands of bacteria and archae have now been screened for their presence. Analysis of sequenced prokaryotic genomes indicates that they are common--all free-living bacteria and archaea appear to code for them. Restriction enzymes are exceedingly varied; they range in size from the diminutive Pvu II (157 amino acids) to the giant Cje I (1250 amino acids) and beyond. Among over 3,000 activities that have been purified and characterized, more than 250 different sequence-specificities have been discovered. Of these, over 30% were discovered and characterized at New England Biolabs.

3. The search for new specificities continues, both biochemically, by the analysis of cell-extracts, and computationally, by the analysis of sequenced genomes. Although most activities encountered today turn out to be duplicates--isoschizomers--of existing specificities, restriction enzymes with new specificities are found with regularity. Beginning in the early 1980’s, New England Biolabs embarked on a program to clone and overexpress the genes for restriction enzymes. Cloning improves enzyme purity by separating enzymes from contaminating activities present in the same cells. It also improves enzyme yields and greatly simplifies purification, and it provides the genes for sequencing and analysis, and the proteins for x-ray crystallography. • Restriction enzymes protect bacteria from infections by viruses, and it is generally accepted that this is their role in nature. They function as microbial immune systems. When a strain of E.coli lacking a restriction enzyme is infected with a virus, most virus particles can initiate a successful infection. When the same strain contains a restriction enzyme, however, the probability of successful infection plummets. The presence of additional enzymes has a multiplicative effect; a cell with four or five independent restriction enzymes could be virtually impregnable.

4. Restriction enzymes usually occur in combination with one or two modification enzymes (DNA-methyltransferases) that protect the cell’s own DNA from cleavage by the restriction enzyme. • Modification enzymes recognize the same DNA sequence as the restriction enzyme that they accompany, but instead of cleaving the sequence, they methylate one of the bases in each of the DNA strands. The methyl groups protrude into the major groove of DNA at the binding site and prevent the restriction enzyme from acting upon it. • Together, a restriction enzyme and its "cognate" modification enzyme(s) form a restriction-modification (R-M) system. • In some R-M systems the restriction enzyme and the modification enzyme(s) are separate proteins that act independently of each other. • In other systems, the two activities occur as separate subunits, or as separate domains, of a larger, combined, restriction-and-modification enzyme.

5. Restriction enzymes are traditionally classified into three types on the basis of subunit composition, cleavage position, sequence-specificity and cofactor-requirements. However, amino acid sequencing has uncovered extraordinary variety among restriction enzymes and revealed that at the molecular level there are many more than three different kinds. • Type I enzymes are complex, multisubunit, combination restriction-and-modification enzymes that cut DNA at random far from their recognition sequences. Originally thought to be rare, we now know from the analysis of sequenced genomes that they are common. Type I enzymes are of considerable biochemical interest but they have little practical value since they do not produce discrete restriction fragments or distinct gel-banding patterns.

6. Type II enzymes cut DNA at defined positions close to or within their recognition sequences. They produce discrete restriction fragments and distinct gel banding patterns, and they are the only class used in the laboratory for DNA analysis and gene cloning. Rather then forming a single family of related proteins, type II enzymes are a collection of unrelated proteins of many different sorts. Type II enzymes frequently differ so utterly in amino acid sequence from one another, and indeed from every other known protein, that they likely arose independently in the course of evolution rather than diverging from common ancestors. • The most common type II enzymes are those like Hha I, Hind III and Not I that cleave DNA within their recognition sequences. • Enzymes of this kind are the principle ones available commercially. Most recognize DNA sequences that are symmetric because they bind to DNA as homodimers, but a few, (e.g., BbvC I: CCTCAGC) recognize asymmetric DNA sequences because they bind as heterodimers. Some enzymes recognize continuous sequences (e.g., EcoR I: GAATTC) in which the two half-sites of the recognition sequence are adjacent, while others recognize discontinuous sequences (e.g., Bgl I: GCCNNNNNGGC) in which the half-sites are separated. Cleavage leaves a 3´-hydroxyl on one side of each cut and a 5´-phosphate on the other. • They require only magnesium for activity and the corresponding modification enzymes require only S-adenosylmethionine. They tend to be small, with subunits in the 200–350 amino acid range.

7. The next most common type II enzymes, usually referred to as ‘type IIs" are those like Fok I and Alw I that cleave outside of their recognition sequence to one side. These enzymes are intermediate in size, 400–650 amino acids in length, and they recognize sequences that are continuous and asymmetric. They comprise two distinct domains, one for DNA binding, the other for DNA cleavage. They are thought to bind to DNA as monomers for the most part, but to cleave DNA cooperatively, through dimerization of the cleavage domains of adjacent enzyme molecules. For this reason, some type IIs enzymes are much more active on DNA molecules that contain multiple recognition sites. • The third major kind of type II enzyme, more properly referred to as "type IV" are large, combination restriction-and-modification enzymes, 850–1250 amino acids in length, in which the two enzymatic activities reside in the same protein chain. These enzymes cleave outside of their recognition sequences; those that recognize continuous sequences (e.g., Eco57 I: CTGAAG) cleave on just one side; those that recognize discontinuous sequences (e.g., Bcg I: CGANNNNNNTGC) cleave on both sides releasing a small fragment containing the recognition sequence. The amino acid sequences of these enzymes are varied but their organization are consistent. They comprise an N-terminal DNA-cleavage domain joined to a DNA-modification domain and one or two DNA sequence-specificity domains forming the C-terminus, or present as a separate subunit. When these enzymes bind to their substrates, they switch into either restriction mode to cleave the DNA, or modification mode to methylate it.

8. Type III enzymes • Type III enzymes are also large combination restriction-and-modification enzymes. They cleave outside of their recognition sequences and require two such sequences in opposite orientations within the same DNA molecule to accomplish cleavage; they rarely give complete digests. No laboratory uses have been devised for them, and none are available commercially.

9. 一．实验目的及背景 • 核酸限制性内切酶是一类能识别双链ＤＮＡ中特定碱基顺序的核酸水解酶，这些酶都是从原核生物中发现，它们的功能犹似高等功物的免疫系统， 用于抗击外来ＤＮＡ的侵袭。 • 限制性内切酶以内切方式水解核酸链中的磷酸二酯键， 产生的ＤＮＡ片段５’端为Ｐ，３’端为ＯＨ。

10. 限制酶的类型 • 根据限制酶的识别切割特性， 催化条件及是否具有修饰酶活性可分为Ⅰ、Ⅱ、Ⅲ型三大类。 • Ⅰ类和Ⅲ类限制性内切酶，在同一蛋白分子中兼有甲基化作用及依赖ATP的限制性内切酶活性。 • Ⅰ类限制性内切酶结合于特定识别位点，且没有特定的切割位点，酶对其识别位点进行随机切割，很难形成稳定的特异性切割末端。 • Ⅲ类限制性内切酶在识别位点上切割，然后从底物上解离下来。 • 故Ⅰ类和Ⅲ类酶在基因工程中基本不用。

11. Ⅱ型酶 • Ⅱ型酶就是通常指的ＤＮＡ限制性内切酶. • 它们能识别双链ＤＮＡ的特异顺序，并在这个顺序内进行切割，产生特异的ＤＮＡ片段； • Ⅱ型酶分子量较小，仅需Mg2+作为催化反应的辅助因子，识别顺序一般为４～６个碱基对的反转重复顺序； • Ⅱ型内切酶切割双链ＤＮＡ产生３种不同的切口－－５’端突出；３’端突出和平末端。 • 正是得益于限制性的内切酶的发现和应用， 才使得人们能在体外有目的地对遗传物质ＤＮＡ进行改造，从而极大地推动了分子生物学的兴旺和发展。

12. 酶切反应中应注意以下几个问题： １．内切酶： • 不应混有其它杂蛋白特别是其它内切酶或外切酶的污染； • 注意内切酶的识别位点及形成的粘性末端； • 内切酶的用量 根据内切酶单位和ＤＮＡ用量而定，通常 1u指在适当条件下，1小时内完全酶解１ug特定ＤＮＡ底物所需要的限制性内切酶量，使用中一般以１ug DNA对２－３u酶短时间为宜。 • 同时内切酶体积不能超过反应体系１０％，因内切酶中含５０％甘油，体系中甘油超过５％会抑制内切酶活力； • 内切酶操作应在低温下进行（冰上）；使用时防止操作中对内切酶的污染。

13. ２．ＤＮＡ： • 作为内切酶底物，ＤＮＡ应该具备一定的纯度，其溶液中不能含酚、氯仿、乙醚、SDS、EDTA、高盐浓度、酒精等，这些因素的存在均不同程度影响限制性内切酶的活力。 • 这种抑制可通过： • 增加酶作用单位数（10~20U/ug DNA）、 • 增大反应体积以稀释可能的抑制剂 • 或延长反应时间加以克服。

14. ３．反应缓冲液： • 反应缓冲液主要由Tris·HCl、NaCl、Mg2+组成，其中Mg2+为内切酶辅基； • Tris·HCl维持反应体系pH值在7.2-7.6之间； • NaCl浓度不同形成３种级别的离子强度： • 低盐（10mM NaCl) • 中盐（50mM NaCl） • 高盐（100mM NaCl） • 不同的内切酶选择特定的反应缓冲液。

15. ４．酶解温度与时间： • 大多数限制酶反应温度为３７℃，如EcoRⅠ, HindⅢ, BamHⅠ, PstⅠ等，也有如BclⅠ需在５０℃下进行反应， • 反应时间根据酶的单位与ＤＮＡ用量之比来定，原则是酶：ＤＮＡ=２－３：１ • ２小时即可，充分酶解。

16. 二、实验试剂 • 限制性内切酶 • ＤＮＡ（λＤＮＡ和质粒ＤＮＡ） • １０×buffer:50mM Tris HCl pH7.5 • 100mM NaCl • 10mM MgCl2 • 无菌水

17. 三．实验方法 • １．按下表分别加入各试剂（注意限制性内切酶最后加入且在冰上操作）于Eppendorf管中。 • DNA １μg • 10×buffer 2.5μl • 无菌水 • 内切酶 2μ • 总体积： 25μl

18. ２．将反应体系充分混匀，并于台式离心机上短暂离心。２．将反应体系充分混匀，并于台式离心机上短暂离心。 • ３．Eppendorf管封上封口膜于３７℃水管中反应２小时。 • ４．反应结束后加入EDTA至终浓度10mM终止反应。 • ５．取１０μl反应液加２μl Loading buffer混匀于１％琼脂糖凝胶上４０伏电泳２－３小时。 • ６．紫外透射仪上检查实验结果。

19. 四．结果与分析 • １．记录紫外透射仪上观察到的结果。 • ２．分析实验结果的成因。 • 问题与讨论： • １．在整个酶切反应过程中应注意哪些问题？ • ２．如何选择ＤＮＡ和限制性内切酶的用量？ • 反应体系中为何内切酶用量不能超过整个反应体系的１０％？